Proc. 14th INA Conf., Reston VA, USA (Guest Ed. J. Self-Trail)
J. Nannoplankton Res. 35 (2), 2015, pp.101-110 © 2015 International Nannoplankton Association
ISSN 1210-8049 Printed by The Sheridan Press, USA

101

An improved sample preparation technique for calcareous
nannofossils in organic-rich mudstones
Jamie L. Shamrock*, Eric J. Muñoz, John H. Carter

ExxonMobil Exploration, 22777 Springwoods Village Parkway, Spring, TX 77067, USA; *jamie.l.shamrock@exxonmobil.com
Manuscript received 19th April, 2015; revised manuscript accepted 6th May, 2015

Abstract Calcareous nannofossils have been routinely applied as a biostratigraphic tool in the oil industry formany decades. Industrial samples are traditionally prepared using a slurry-smear technique, which is relatively fast,
simple and uses no chemicals. Recent increases in unconventional resource plays in the oil industry have resulted ina significant number of nannofossil biostratigraphy projects derived from organic-rich source-rocks. While slurrysmear slides are satisfactory for nannofossil samples in most lithologies, this preparation technique has proved insufficient for such samples. Shale-gas source-rocks are often rich in clays, silts, opaque minerals and organic components.
In addition, amorphous organic material in the sediment can bond grains together, trapping nannofossil specimens
and impeding identification. We propose a modified method of sample preparation for calcareous nannofossils thatminimises these issues and increases the number of identifiable specimens. This relatively simple method is modeled
after foraminifera cleaning techniques for palaeothermometry, which focus on minimising adulteration of fossil specimens. The proposed technique for calcareous nannofossils uses sodium hypochlorite to lighten the organic matter andto aid in the dissociation of nannofossils from background sediments. Semi-quantitative analysis shows significant
reduction in organic matter, and increases in relative abundance and species richness. The technique was applied tosamples from the Upper Cretaceous Eagle Ford and La Luna Formations and the Upper Jurassic-Lower Cretaceous
Haynesville Formation. Overall, the method is relatively quick, and minor increases in sample preparation time areoffset by improved data quality and potential for higher-resolution biostratigraphy in unconventional resource plays.
Keywords calcareous nannofossils, sample preparation, sample processing, organic-rich mudstones, unconventional hydrocarbon resources

1. Introduction
Calcareous nannofossils are the remains of coccolithophores (photosynthetic, unicellular algae of Division Haptophyta), which are generally high in abundance and diversity in open-marine settings. The biostratigraphic utility ofthese microfossils has been significant in both industry andacademia since the 1960s, and they continue to be a key
component in biostratigraphic analysis. Industrial calcareous nannofossil samples (usually in the form of cuttings)
are commonly prepared as slurry-smear slides (following
Watkins & Bergen, 2003). This method is relatively fastand simple, and uses only dilute (10%) hydrochloric acid(HCl) for cleaning glassware between samples, yielding
excellent results in most lithologies (Plate 1).
Recently, the hydrocarbon industry has developed assets in unconventional resources, such as shale-gas plays(Holditch, 2003; Boughal, 2008; Stevens & Kuushraa,
2009; Toon, 2011; Leimkuhler & Leveille, 2012). Lithologies from shale-gas plays tend to be high in clays, opaqueminerals, such as pyrite and marine-derived amorphousorganic matter (AOM), with intermittent terrestrial organics. When prepared using the slurry-smear method, thesecomponents significantly obscure individual nannofossil
specimens and can cause aggregations of nannofossils andbackground sediments (Plate 1). This impedes specieslevel identification of nannofossil specimens and can skew
abundance and diversity data. With an increasing numberof industry-based biostratigraphy projects derived fromshale-gas assets, we address the need for a modified nannofossil sample preparation technique that both lightens
organic matter and dissociates specimens from AOM andclays, without the dissolution of carbonate or the mechanical degradation of nannofossil specimens.
2. Methodology
2.1 Experimentation
The sample preparation method presented here is based on
foraminifera cleaning procedures for palaeothermometry,
which are designed to remove clays and organic material without altering either morphology or isotopic ratios.
Such procedures are often of extended duration (Bice &Norris, 2005; Blanco-Ameijeiras et al., 2012), going togreat lengths to ensure that dissolution of fossil carbonate
is minimised. The goal of this study was to modify suchpreparation procedures for the needs of nannofossil identification using basic light microscopy at ~1000–1250x
magnification.
Several cleaning procedures were compared (Ander-

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Plate 1Comparison of slurry-smear slide preparations of (A) carbonate-dominated vs. (B) organic-rich lithologies
Organic matter particularly evident in Eagle Ford Fm in PPL (lower right)
Photomicrographs taken at 630x magniﬁcation for wider FOV

XPL

XPL

open-marine chalk, Swanage, southern England

restricted-marine organic-rich mudstone, Eagle Ford Formation, Texas, USA

son, 1961; Clark, 1973; Hodgkinson, 1991; Martin & Lea,
2002; Barker et al., 2003; Bice & Norris, 2005; Weldeab etal., 2006; Krause-Nehring et al., 2011; Blanco-Ameijeiras
et al., 2012) and were considered for both the base procedure and potential modifications. Our final procedure uses
sodium hypochlorite (NaClO - bleach), as it was noted forits superior cleaning ability and relatively low potential forreactivity (Anderson, 1961; Hodgkinson, 1991; Gaffey &Bronnimann, 1993; Pingitore et al., 1993; Bice & Norris,2005). We eliminated a buffered hydrogen peroxide (H2O2)
solution as the primary reagent, because of its potential
for reaction with organic matter and production of formic
acid, which can dissolve carbonate fossils (Anderson,
1961; Hodgkinson, 1991; Gaffey & Bronnimann, 1993).

PPL

PPL

In addition, some studies have shown hydrogen peroxide
to be less effective than bleach in the lightening of organic
matter (Hodgkinson, 1991; Bice & Norris, 2005).
The basic cleaning procedure was tested at 9.0, 9.5
and ~12.0pH (pure household bleach). For each pH level,samples were exposed to one, two or three iterations of
the cleaning procedure. Although initial trials showed anotable reduction of organic matter at 9.0 and 9.5pH, significant amounts remained in the final slide preparations.
Bice & Norris (2005) achieved exceptional results using
pure bleach (5.25-6.0% NaClO by volume), so this moreaggressive approach was included in later trials. Thoughinitial readings of pure bleach are notably basic (~11.512.0pH), the pH decays rapidly through the reaction with

Preparation...organic-rich mudstones

organic matter at elevated temperatures, stabilising between 9.0-9.5pH.
In addition to variations in pH and number of cleaning
iterations, samples were compared with and without theuse of an ultrasonic bath. It is suspected that ultrasonicpressure may damage microfossils (Hodgkinson, 1991).
Research by Clark (1973) detailed such effects, but also
identified an ideal time/pressure relationship for suchcleaning procedures. This requires an ultrasonic bath capable of changes in frequency. While we acknowledge
the potential for ultrasonic cleaning in nannofossil preparation, particularly in more lithified samples, we saw nosignificant difference in our samples prepared with andwithout an ultrasonic bath. We conclude that ideal results
can be achieved without this process.
The final procedure (detailed below) is a modification of Bice & Norris (2005) and Anderson (1961). A primary goal was to identify maximum effectiveness withminimal processing, and to decrease potential damage tofossil specimens and subsequent data. Optimum results
were achieved with two iterations of the bleach-cleaning/bicarbonate-washing procedure and no ultrasonic bath.
The best results were achieved using pure bleach. Cloroxbrand bleach is recommended, as it has the highest NaClO
by volume (6%) and, therefore, stronger cleaning potential. Duration of washing is based on Anderson (1961).
The bleaching is followed by washing with a bicarbonate
solution to disperse the sample. Thorough washing with
deionised water is critical after applying the bicarbonate
solution, as detailed in Step 3, below. We found that insufficient washing results in residual bicarbonate ions, whichrecrystallise on drying, obscuring a significant proportionof the coverslip. With batch-processing, this should not inhibit preparation efficiency. Finally, use of the centrifuge
separates the sample into two distinct layers. We preparedslides from both layers, as well as from mixed samples.
We consistently found optimum nannofossil concentrations in the lower (darker) layer, while the upper (lighter)layer is dominated by clays. Until further investigation, wesuggest preparation of two slides, one from each layer, tominimise potential issues with size fractionation.
2.2 Resultant procedure
Preparation: Place ~0.1g of sample into a 15ml glass centrifuge tube. Fill a beaker with ~5-6cm of water and place
on a hot-plate. Steps 1-3 must be conducted under a fume

103

hood.
Step 1 - NaClO wash: Add 3ml pure bleach to the sample,
then place the centrifuge tube on a Vortex mixer at medium speed for 3-5 seconds to ensure good contact between
sediment and reagent. Boil the sample in a water-bath for
15 minutes. Centrifuge the sample at 1000rpm for 7 minutes, then decant the supernatant. Repeat Step 1.
Step 2 - NaHCO3 wash: Add ~5ml of 9.5pH sodium bicarbonate solution to the sample, then place the centrifuge
tube on a Vortex mixer at medium speed for 3-5 seconds.
Boil the sample in a water-bath for 15 minutes. Centrifuge
at 1000rpm for 7 minutes, then decant the supernatant. Repeat Step 2, but instead, centrifuge the sample at 2500rpm
for 10 minutes.
Step 3 - H2O wash: Add 5 ml deionised water to the sample, then place the centrifuge tube on a Vortex mixer at
medium speed for 5-10 seconds. Centrifuge the sample at
2000rpm for 4 minutes, then decant the supernatant. Repeat this process for a total of 3 iterations. After the final(third) decanting, add 5ml of deionised water and place
the centrifuge tube on a Vortex mixer at low speed to mix
overnight, if possible, or for a minimum of 4 hours. Centrifuge at 2000rpm for 10 minutes, then decant the supernatant.
Step 4 - slide preparation: After the final centrifugation,
the sample will be separated into two layers. Prepare aslide from each layer, using the method of Watkins & Bergen (2003). If dispersion of sediment across the coverslip
is sufficient on initial application, a double-slurry preparation may not be necessary. Take care not to overload thecoverslip with sediment. Optimum thickness can often be
judged by placing the coverslip on a sheet of white paper; ideally, the coverslip should look very slightly dusty,
though it should be noted that sediment colour can vary.
Most centrifuge and Vortex machines can hold six ormore samples, which allows for batch processing. Using
two six-tube centrifuge machines and a 12-tube Vortex attachment, we were able to process two 12-sample batchesa day (24 samples, 48 slides) using the above procedure.
3. Results
Samples were selected from three well-known, unconventional-resource formations: 1) the Cretaceous Eagle Ford
Formation (Fm), 2) the Cretaceous La Luna Fm and 3)
the Jurassic-Lower Cretaceous Haynesville Fm (Figure 1).
In general, the depositional settings for these formations

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Shamrock, Muñoz, Carter

grains, land-derived phytoclasts, AOM and opaque minerals. Nannofossil specimens from untreated samples can bedifficult to identify to species level, and meaningful abundance and diversity counts can be compromised. Here, we
compare results for residual organic content and for abundance and species richness of nannofossil assemblages forseveral of the preparation variations discussed above.

Figure 1: Map of North and South America, illustrating generalised
distributions of Jurassic Haynesville and Cretaceous Eagle Ford and La
Luna Formations

provide for both a shelf/platform carbonate component, as
well as a fluvial/deltaic siliciclastic component (Rangel etal., 2000; Hammes et al., 2011; Driskill et al., 2012). As aresult, these formations show varying proportions of carbonate and siliciclastic material, as well as variations interrestrial and marine organic matter. The palaeotopography provided a restricted to semi-restricted marine setting,with water-column stratification and dysoxic to anoxic
bottom-water conditions (Rangel et al., 2000; Hammes etal., 2011; Driskill et al., 2012). These conditions promoted
the preservation of organic material, as well as the frequent formation of pyrite. Total organic carbon (TOC) of
these formations ranges from 0.5 to 6.9% (average ~2.04.5%; Zumberge, 1984; Hammes & Carr, 2009; Dawson& Almon, 2010).
Many of the backgroundcomponents in these lithologies hinder nannofossil identification by both obscuring
individual specimens and byclumping multiple specimens
together with other particles,
such as carbonate and quartz
Figure 2: Illustration of average residual organics per FOV after various treatment procedures. Estimates
based on Compton (1962) and Reid
(1985). N = 50 FOV at 630x magnification in PPL. Ideal results (<1%residual organics/FOV) achieved
with 2-3 iterations of cleaning/
washing procedure

3.1 Residual organics
Residual organics were compared for each sample undera variety of treatments, including variations in pH, thenumber of wash iterations, and with and without the useof an ultrasonic bath. The relative abundance of residualorganics was estimated using Compton (1962) for abundances of 2-50% per field of view (FOV) and Reid (1985)
for abundances of 1% or less per FOV. Estimates weremade at 630x magnification in plane polarised light (PPL),
where N = 50 FOV. Results are summarised in Figure 2
and Table 1.
In untreated slurry-smear slides, organic material comprised an average of ~7-14% of the FOV. Treatments at
9.5pH showed a reduction in organic material (µ = ~49%); however, significant organic material remained,even with three iterations of the treatment. Pure bleach
was most successful in lightening and dispersing organic
matter. A single iteration of the treatment with pure bleachgives results similar to 9.5pH after three treatments (Figure 2). Optimum results were achieved with two and three
iterations, both consistently giving an average organicPreparation...organic-rich mudstones

Table 1: Residual organic matter per FOV after various treatments. Estimates based on Compton (1962) and Reid (1985). N = 50 FOV at 630x
magnification in PPL

matter content of <1% per FOV (N = 50 FOV). Ideally,
samples should undergo as little processing as possible,
with two iterations sufficient to achieve the desired results.
Results for each formation are summarised in Table 1. Decreasing background organic matter, increasing sediment
dispersion and cleaner individual specimens are illustrated
in Plates 2-4 for the Eagle Ford, La Luna and HaynesvilleFms, respectively. Remaining background material obscuring nannofossil specimens is predominantly limited toopaque minerals. Photomicrographs are all taken at 630x
magnification in cross-polarised light (XPL, left) and PPL(right).
3.2 Nannofossil abundance andspecies richness
One of the greatest challenges of working with untreatedsamples from organic-richmudstones is the species-level identification of obscured
specimens. Many species reFigure 3: Abundance data for Eagle Ford, La Luna and HaynesvilleFms, showing relative changes inproportion of unidentifiable vs.
identifiable nannofossil specimens, and total nannofossil abundance after varying sample treatments. Unidentifiable specimens
refer to those obscured by organic
matter or those adhering to background sediment, and does notinclude broken specimens withincomplete morphology

105

quire an unobscured view of the central-area structure forpositive identification. Subsequently, numerous specimens
in an untreated sample may be labeled ‘unidentifiable’. In
addition, aggregates of multiple specimens, along withother background components, result in tentative relative
abundance estimates.
In this study, nannofossil relative abundance and species richness is based on one full traverse at 1000x (~250
FOV). Two additional traverses were completed to scan
for rare taxa, which were given a value of one. As the main
goal of this portion of the study was to compare changes
in relative abundance and species richness in one sample
under a variety of preparations, care was taken to makeall slides to a consistent thickness. We noted a significant
increase in the number of identifiable specimens in treatedsamples (Figure 3). In addition, we observed an increasein species richness, likely due to improved visibility andgreater confidence in species-level identification (Table 2).
Treated samples also showed a modest increase in totalnannofossil abundance, either due to an underestimationof the number contained in untreated aggregates, or due toa slight concentration of nannofossils in samples treatedfor organics and clays (Figure 3, Table 2).
3.3 Cost/beneﬁt
Although there is a slight increase in processing time, thiscost is significantly offset by the increased quality of thesample preparations. This reduces total microscope time

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Shamrock, Muñoz, Carter

Plate 2Comparison of sample Eagle Ford 1 with (A) untreated slurry-smear slide, (B) 9.5pH, 3 iterations, no sonic and (C)pure bleach, 2 iterations, no sonicOrganic matter particularly evident in PPL. Selected specimens highlighted for comparison
Photomicrographs taken at 630x magniﬁcation for wider FOV

PPL

XPL
PPL

XPL
PPL

XPL

needed for analysis and produces biostratigraphic datawith greater accuracy. In addition, the procedure uses
household bleach as the primary reagent, which is mild
enough to permit use of the procedure on drilling rigs, andis inexpensive.

4. Conclusions
We have presented a method of calcareous nannofossil
sample preparation that lightens organic matter and increases liberation of individual nannofossil specimensfrom background sediment in organic-rich mudstones.

Preparation...organic-rich mudstones

107

Plate 3Comparison of sample La Luna 1 with (A) untreated slurry-smear slide, (B) 9.5pH, 3 iterations, no sonic and (C) purebleach, 2 iterations, no sonicOrganic matter particularly evident in PPL. Selected specimens highlighted for comparison
Photomicrographs taken at 630x magniﬁcation for wider FOV

PPL

XPL
PPL

XPL
PPL

XPL

This procedure is modeled after foraminifer cleaning techniques, but was modified to be sufficient for basic lightmicroscopy at ~1000x magnification. Through numerous
revisions and tests of the process, we have detailed a rela-

tively quick and simple procedure that maximises nannofossil recovery and identification with minimal processing.
This pretreatment uses pure bleach (NaClO ~5.25-6.0%
by volume), followed by washes with bicarbonate solu-

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Shamrock, Muñoz, Carter

Plate 4Comparison of sample Haynesville 2 with (A) untreated slurry-smear slide, (B) 9.5pH, 3 iterations, no sonic and (C)pure bleach, 2 iterations, no sonicOrganic matter particularly evident in PPL. Selected specimens highlighted for comparison
Photomicrographs taken at 630x magniﬁcation for wider FOV

PPL

XPL
PPL

XPL
PPL

XPL

tion and deionised water. Up to 24 nannofossil samples
can be prepared per day with batch processing. We believe
the procedure provides significantly improved data anddecreases total microscope time for a relatively modest in-crease in processing time and cost of laboratory supplies.
With increased assets in unconventional resources, agreater number of calcareous nannofossil biostratigraphy
projects are derived from organic-rich mudstones. WhilePreparation...organic-rich mudstones

109

Barker, S., Greaves, M. & Elderfield, H. 2003. A study of cleaning procedures used for foraminiferal Mg/Ca paleothermom-etry. Geochemistry, Geophysics, Geosystems, 4(9): 8407,
doi: 10.1029/2003GC000559.

Bice, K.L. & Norris, R.D. 2005. Data report: stable isotope ratios

of foraminifers from ODP Leg 207, Sites 1257, 1258, and1260, and a cleaning procedure for foraminifers in organ-

ic-rich shale. In: D.C. Mosher, J. Erbacher & M.J. Malone(Eds). Proceedings of the ODP, Scientific Results, 207: 1-23
[Online]http://www-odp.tamu.edu/publications/207_SR/

VOLUME/CHAPTERS/104.PDF.

Blanco-Ameijeiras, S., Lebrato, M., Stoll, H.M., Inglesias-Rodriguez, M.D., Méndez-Vicente, A., Sett, S., Müller, M.N.,
Oschlies, A. & Shultz, K.G. 2012. Removal of organic mag-nesium in coccolithophore calcite. Geochimica et Cosmochimica Acta, 89: 226-229.

Boughal, K. 2008. Unconventional plays grow after Barnett
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Clark, D.F. 1973. Effects of Ultrasonic Pressure on Calcareous
Table 2: Detail of relative abundance data for Eagle Ford, La Luna andHaynesville Fms, showing changes in relative proportions of unidentifiable versus identifiable nannofossil specimens, as well as total nannofossil abundance and species richness after varying sample treatments

traditional methods of industrial sample preparation are
excellent for most lithologies, they are insufficient for
sediments rich in marine and terrestrial organic matter.
We document significant decreases in organic matter, andconcomitant increases in both the number of identifiable
specimens and species richness, in all treated samples.
This methodology can increase species-level identification, improve biostratigraphic data and significantly raise
confidence in biostratigraphic interpretations.

Acknowledgements

The authors would like to thank Karen M. Love and M. ShaneLong (ExxonMobil) for permission and encouragement to workon the project, as well as Ellen Seefelt (US Geological Survey),
David Jutson (BP) and the ExxonMobil Biostratigraphy CoreTeam for their reviews and suggestions.

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